Enzyme inhibitors for metabolic redirection

ABSTRACT

A method is described for improving the pharmacokinetics of a drug in a subject, by co-administering oligomers, preferably PMO&#39;s (phosphorodiamidate morpholino oligonucleotides), antisense to RNAs encoding drug-metabolizing enzymes, particularly p450 enzymes. The oligomers reduce production of the drug-metabolizing enzymes, which extends drug half-life and effectiveness and/or decreases drug toxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/802,859, filed Feb. 19, 1997, now abandoned which is a continuationof commonly owned U.S. provisional application Ser. No. 60/012,219,filed Feb. 23, 1996, both of which are incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to methods of improving the performance ofdrugs which are metabolized by p450 enzymes, by antisense inhibition ofthe particular enzyme. Typically, the p450 enzyme is induced by anexogenous substance or by the drug itself.

BACKGROUND OF THE INVENTION

When a drug is introduced to a biological system, multiplepharmacokinetic processes begin to affect the ultimate efficiency of thedrug, determining how rapidly, in what concentration, and for how longthe drug will be available to the target organ. In general, lipophilicxenobiotics are metabolized to more polar and hence more readilyexcretable products. The role metabolism plays in the inactivation oflipid soluble drugs can be quite dramatic. For example, lipophilicbarbiturates such as thiopental and phenobarbital would have extremelylong half-lives were it not for their metabolic conversion to more watersoluble compounds. Many potential anticancer drugs are deemedunbeneficial because their half-life is too brief to achieve any usefultherapeutic effect.

The metabolic conversion of an ingested compound (such as a drug or afood additive) into a form which is readily cleared from the body istermed biotransformation or detoxification. Compounds ingested byorganisms are generally biotransformed in two phases. In Phase 1, termedfunctionalization, a reactive site, such as an amine, thiol, or hydroxylgroup, is introduced, generally via an oxidation reaction. In Phase 2,termed conjugation, a water-soluble group is added to the reactive site.Phase 2 typically involves addition of a glucuronic acid, saufuric acid,acetic acid or amino acid to the compound.

Phase 1 reactions are frequently catalyzed by the cytochrome p450superfamily of enzymes. In a typical Phase 1 reaction, a cytochrome p450enzyme uses oxygen and NADH to add a reactive group, such as a hydroxylradical, to a drug. The reactive intermediates produced may be much moretoxic than the parent molecule, and may cause damage to proteins, RNA,and DNA within the cell (Vermeulen, N. P. E., “Role of metabolism inchemical toxicity,” in: Ioannides, C., ed., Cytochrome p450: Metabolicand Toxicological Aspects. Boca Raton, Fla.: CRC Press, Inc; 1996, pp29-53).

Phase 2 conjugation reactions, which generally follow Phase 1 activationreactions, often reduce the toxicity of reactive intermediates formed byPhase 1 reactions. Phase 2 conjugation transforms the drug into awater-soluble compound that can be excreted, e.g. through urine or bile.Several types of conjugation reactions occur in the body, includingglucuronidation, sulfation, and glutathione and amino acid conjugation.In some instances, the parent drug may already possess a functionalgroup that forms a conjugate directly. For example, the hydrazide moietyof isoniazide is known to form an acetyl conjugate in a Phase 2reaction. This conjugate is then a substrate for a Phase 1 typereaction, namely, hydrolysis to isonicotinic acid. Thus, Phase 2reactions may in some instances actually precede Phase 1 reactions.

Correlations have been noted between altered Phase 1 and/or Phase 2metabolic activities and increased risk of diseases such as cancers andliver disease, and in adverse drug responses. For example, some drugs(such as acetaminophen) are metabolically converted to reactiveintermediates that are toxic to various organs. These toxic reactionsmay not be apparent at low drug dosages, when subsequent steps oralternative pathways are not overwhelmed or compromised and theavailability of endogenous co-substrates (glutathione, glucuronic acid,sulfate) is not limited. When these resources are exhausted, however,the toxic pathway may prevail, resulting in overt organ toxicity orcarcinogenesis.

Many drugs and other xenobiotic agents are capable of inducing geneswhich encode drug-metabolic enzymes, enhancing the levels of theseenzymes and, consequently, accelerating the metabolic reactionscatalyzed by these enzymes. Such accelerated metabolism may cause adecrease in the half-life and pharmacologic efficacy of the substratedrug. Induction genes encoding drug-metabolizing compounds couldexacerbate drug-mediated tissue toxicity by increasing steady-statelevels of reactive or toxic intermediates.

A need exists in the art for modulating the pharmacokinetics of variousdrugs in patients. The present invention achieves this by decreasing theproduction of one or more specific drug-metabolizing enzymes which areinduced, either by the drug itself or by another xenobiotic agent towhich the patients have been exposed. Decreased drug metabolism resultsin an increased drug half-life. The dosage of the drug can then bereduced, since the lower dose has equivalent bioavailability to that ofa higher dose in the absence of such modulation, and toxicitiesassociated with high drug dosage can be circumvented. Reducing theavailability of metabolically toxic pathways thus increases the safetyof the drug.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of improving thepharmacokinetics of a drug administered to a subject, where the drug isknown to be metabolized in vivo by a cytochrome p450 enzyme that reducesthe effectiveness of the drug. In accordance with the method, oneco-administers with the drug a morpholino antisense oligomer effectiveto reduce synthesis of the drug-metabolizing cytochrome p450 enzyme, byhybridizing to a target RNA molecule which encodes the enzyme. Inpreferred embodiments of the method, the drug itself induces thedrug-metabolizing p450 enzyme, or the subject has been exposed to axenobiotic agent which induces such an enzyme.

In one embodiment, the antisense oligomer hybridizes to a region of thetarget RNA molecule which includes the AUG translation start site. Inanother embodiment, the target RNA molecule is pre-mRNA, and theantisense oligomer hybridizes to a region of the pre-mRNA which includesan intron-exon boundary or an exon-intron boundary.

Preferably, the antisense oligomer is at least 15 nucleotides in length.Preferred oligomers are morpholino oligomers having an unchargedbackbone comprising phosphoramidate or, preferably, phosphorodiamidatelinkages. The antisense oligomer preferably hybridizes to a region ofthe target RNA with a T_(m) greater than 37° C. The sequence of theoligonucleotide can be one selected from the group consisting of SEQ IDNOs: 16-35 and 46-47, preferably from SEQ ID NOs: 26-35 and 4647(targeted to human RNA sequences), and more preferably from SEQ ID NOs:27, 30, 34, 35, and 46-47.

The targeted cytochrome p450 enzyme is preferably selected from thegroup consisting of CYP1A1, CYP1A2, CYP2A6, CYP2B1, CYP2C9, CYP2C19,CYP2D6, CYP2E1, CYP3A2, CYP3A4, and CYP6A1 enzymes. In a preferredembodiment, where the subject is a human subject, the cytochrome p450 ispreferably selected from the group consisting of CYP1A1, CYP1A2, CYP2A6,CYP2B1, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 enzymes, and morepreferably from the group consisting of CYP1A2, CYP2B1, CYP2E1, andCYP3A4 enzymes.

In selected embodiments, the enzyme is CYP2E1, and the drug isacetaminophen, or the enzyme is from the CYP2B or CYP3A subfamily,preferably CYP2B1, and the drug is phenobarbital or hexobarbital. Infurther embodiments, the enzyme is CYP3A4, and the drug is an antibioticselected from the group consisting of clarithromycin, erythromycin,rifampicin, rifampin, rifabutin, and rapamycin; or the enzyme is CYP3A4or CYP1A2, and the drug contains an estrogen or estradiol. In stillfurther embodiments, the enzyme is CYP3A4, the drug is a proteaseinhibitor or a non-nucleoside reverse transcriptase inhibitor, and theinducing xenobiotic is a CYP3A4-inducing non-nucleoside reversetranscriptase inhibitor.

In a preferred embodiment, the antisense oligomer is administered orallyto the subject, typically in an amount of at least 1 mg/kg body weight.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the steps in oxidation of a drug by a cytochrome p450;

FIG. 2 shows alternative pathways for acetaminophen metabolism, leadingto a toxic product or to a non-toxic mercapturate conjugate, whereGSH=glutathione and AC*=reactive intermediate;

FIG. 3 depicts the results of ELISA for Cytochrome p450 2B1 (CYP2B1)from liver microsomes treated with various antisense oligonucleotides(see Example 1), in the presence or absence of the CYP2B1 inducing agentphenobarbital (PB);

FIG. 4 depicts the levels of CYP2E1 isozyme from cultures treated withincreasing amounts of the antisense oligonucleotide 2E1-1560 (SEQ ID NO:19), assayed by ELISA for comparative amounts of the CYP2E1 isozymepresent (see Example 2);

FIGS. 5A-5E shows several preferred subunits having 5-atom (A), six-atom(B) and seven-atom (C-E) linking groups suitable for forming polymers;

FIGS. 6A-A to 6E-E show the repeating subunit segment of exemplarymorpholino oligonucleotides, designated A—A through E—E, constructedusing subunits A-E, respectively, of FIG. 5; and

FIG. 7 shows a Western blot of liver microsome samples comparingrelative levels of CYP3A2 isozyme in rats injected i.p. with saline(lane 1), or with 15 nmoles of CYP3A2 antisense PMO, SEQ ID NO: 25(lanes 2 and 3), or orally administered 60 nmoles of CYP3A2 antisensePMO (lanes 4 and 5), 24 hours prior to organ harvesting, where the laneslabeled “NADPH Reductase” are a control for total protein on the blot.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

A “xenobiotic” is a chemical substance which is foreign to a biologicalsystem. Xenobiotics include: naturally occurring compounds which areforeign, i.e., non-native, to the biological system in question, drugs,environmental agents, carcinogens, and insecticides.

A “drug” refers to a chemical substance administered to an animal for atherapeutic purpose. Such agents may take the form of ions, smallorganic molecules, peptides, proteins or polypeptides, oligonucleotides,and oligosaccharides, for example. The agent is typically administeredto cause an observable and desirable change in the structure, function,or composition of a cell upon uptake by the cell. Such changes include,for example, increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, or increased or decreased cell proliferation.

“Induction” of a gene refers to the switching on or enhancement ofexpression of the gene by a stimulus such as an inducer molecule, e.g. ahormone or exogenous substance, or by another stimulus such as heat. Inthe context of the present invention, induction by an exogenoussubstance (xenobiotic) is typically intended. Induction of an enzymetypically results from induction of the gene encoding the enzyme.

A “nuclease-resistant” oligomeric molecule (oligomer) is one whosebackbone is not susceptible to nuclease cleavage. Exemplarynuclease-resistant antisense oligomers are oligonucleotide analogs, suchas phosphorothioate and phosphate-amine DNA (pnDNA), both of which havea charged backbone, and methyl phosphonate, morpholino, and peptidenucleic acid (PNA) analogs, all of which have uncharged backbones.

The terms “antisense oligonucleotide” and “antisense oligomer” are usedinterchangeably and refer to an oligomer having a sequence of nucleotidebases and a subunit-to-subunit backbone that allows the antisenseoligomer to hybridize to a target sequence in an RNA by Watson-Crickbase pairing, to form an RNA:oligomer heteroduplex within the targetsequence. The oligomer may have exact sequence complementarity to thetarget sequence or near complementarity. These antisense oligomers mayblock or inhibit translation of the mRNA containing the target sequence,and/or modify the processing of the mRNA to produce a splice variant ofthe mRNA. Antisense oligonucleotides which are double-stranded DNAbinding agents may inhibit gene transcription.

An oligonucleotide or antisense oligomer “specifically hybridizes” to atarget polynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a T_(m) greater than 37° C., preferablyat least 50° C., and more preferably at least 60° C., 80° C., or higher.Such hybridization preferably corresponds to stringent hybridizationconditions, selected to be about 10° C. lower, and preferably about 5°C. lower than the thermal melting point (Tm) for the specific sequenceat a defined ionic strength and pH. At a given ionic strength and pH,the Tm is the temperature at which 50% of a target sequence hybridizesto a complementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules.

Although the antisense oligomer is not necessarily 100% complementary tothe target sequence, it is effective to stably and specifically bind tothe target sequence such that expression of the target sequence ismodulated. The appropriate length of the oligomer to allow stable,effective binding combined with good specificity is about 8 to 40nucleotide base units, and preferably about 12-25 base units.Mismatches, if present, are less destabilizing toward the end regions ofthe hybrid duplex than in the center. Oligomer bases that allowdegenerate base pairing with target bases are also contemplated,assuming base-pair specificity with the target is maintained.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically hybridizes to a polynucleotide whose sequence is the secondsequence.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligomer with a target RNA sequenceinside a cell even in the presence of many other diverse molecules. Thebase specificity of such binding is sequence specific. For example, asingle-stranded polynucleotide can specifically bind to asingle-stranded polynucleotide that is complementary in sequence.

As used herein, a “morpholino oligomer” or “morpholino oligonucleotide”refers to a polymeric molecule having a backbone which supports basescapable of hydrogen bonding to typical polynucleotides, wherein thepolymer lacks the ribose backbone linked by phosphodiester bonds whichis typical of nucleotides and nucleosides, and instead contains asubunit with a ring nitrogen with coupling through the ring nitrogen. Apreferred “morpholino” oligonucleotide is composed of morpholino subunitstructures of the form shown in FIGS. 5A-E, where (i) the structures arelinked together by phosphorous-containing linkages, one to three atomslong, joining the morpholino nitrogen of one subunit to the 5′ exocycliccarbon of an adjacent subunit, and (ii) Pi and Pj are purine orpyrimidine base-pairing moieties effective to bind, by base-specifichydrogen bonding, to a base in a polynucleotide. Exemplary structuresfor antisense oligonucleotides for use in the invention include themorpholirio subunit types shown in FIGS. 5A-E, with the linkages shownin FIGS. 6A-A to 6E-E. The synthesis, structures, and bindingcharacteristics of morpholino oligomers, including antisense oligomers,are described in detail in co-owned U.S. Pat. Nos. 5,185,444, 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337,all of which are incorporated herein by reference.

A preferred morpholino oligonucleotide is composed of morpholino subunitstructures of the form shown in FIGS. 2B-B, where the structures arelinked together by phosphorodiamidate linkages, joining the morpholinonitrogen of one subunit to the 5′ exocyclic carbon of an adjacentsubunit, Pi and Pj are purine or pyrimidine base-pairing moietieseffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, X═NH₂ or NHR, Y═O, and Z═O, where R is lower alkyl (i.e.C1 to C6, preferably C1 to C4 alkyl). Such structures are described, forexample, in Hudziak et al., Antisense Nucleic Acid Drug Dev. 6, 267-272(1996) and Summerton and Weller, Antisense Nucleic Acid Drug Dev. 7,187-195 (1997). Unless otherwise indicated, “PMO”s referred to hereinhave this structure, where X is NH₂. Also preferred are structureshaving an alternate phosphorodiamidate linkage, where, in FIGS. 2B-B,X═OR, Y═NH or NR, and Z═O.

A “C-5-methyl modified” oligonucleotide is one in which the C-5 hydrogenof cytidine bases has been replaced with a methyl group. A “C-5-propynemodified” or “C-5-propyne pyrimidine-modified” oligonucleotide is one inwhich the C-5 methyl group of thymidine bases and/or the C-5 hydrogen ofcytidine bases has been replaced with a propynyl group (—C≡C—CH₃).

The term “modulating expression” relative to oligonucleotides refers tothe ability of an antisense oligomer to either enhance or reduce theexpression of a given protein by interfering with the expression ortranslation of RNA. In the case of enhanced protein expression, theantisense oligomer may block expression of a suppressor gene, e.g., atumor suppressor gene. In the case of reduced protein expression, theantisense oligomer may directly block expression of a given gene, orcontribute to the accelerated breakdown of the RNA transcribed from thatgene.

An “effective amount” relative to an antisense oligomer refers to theamount of antisense oligomer administered to a mammalian subject, eitheras a single dose or as part of a series of doses, that is effective tospecifically hybridize to all or part of a selected target sequence,thereby reducing expression of the protein encoded by the targetsequence.

“Treatment” of an individual or a cell is any type of intervention in anattempt to alter the natural course of the individual or cell. Treatmentincludes, but is not limited to, administration of a pharmaceuticalcomposition, and may be performed either prophylactically or subsequentto the initiation of a pathologic event or contact with an etiologicagent.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Coadministration” of an antisense oligomer with a drug may beconcurrent with, following, or, preferably, preceding administrationwith the drug, as long as the antisense oligomer is effective tomodulate the metabolism and enhance the efficacy of the drug.

Abbreviations: ON=oligonucleotide; ODN=oligodeoxyribonucleotide; PS orPS-ODN=phosphorothioate oligonucleotide; PMO=phosphoramidate or(preferably) phosphorodiamidate morpholino oligonucleotide

II. Antisense Oligomers

Antisense oligomers effect changes in gene expression (transcription)and protein production (translation) by the complementary hybridizationof relatively short oligonucleotides to single-stranded RNA ordouble-stranded DNA, such that the normal, essential functions of theseintracellular nucleic acids are disrupted. (See, e.g., U.S. Pat. No.5,843,684).

Two mechanisms of action of antisense oligomers on target nucleic acidmolecules have been proposed. In one mechanism, antisense agents arethought to disrupt nucleic acid function via enzymatic cleavage of thetargeted RNA by intracellular RNase H. The oligonucleotide oroligonucleotide analog, which must be of the deoxyribo type, hybridizeswith the targeted RNA, and the duplex activates RNase H to cleave theRNA strand, thus destroying the normal function of the RNA.Phosphorothioate oligonucleotides are a prominent example of antisenseoligomers that operate by this mechanism.

Another mechanism, termed “hybridization arrest”, involves a terminatingevent in which the antisense oligomer binds to the target nucleic acidand thus prevents, by steric hindrance, the binding of essentialproteins, most often ribosomes, to the nucleic acid. Exemplary antisenseoligomers which act by this mechanism include methylphosphonateoligonucleotides and alpha anomer oligonucleotides. (See, e.g., Cohen,Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press,Inc., Boca Raton Fla., 1989.)

The utility of antisense oligomers to modulate the pharmacokinetics ofdrugs or other xenobiotic agents, by decreasing production of specificmetabolic enzymes which are induced by and/or metabolize these agents,requires that the oligomers be amenable to synthesis in largequantities, be taken up by cells and/or transported across cellmembranes, hybridize appropriately to the targeted RNA (i.e., mnRNA orpre-mRNA) and subsequently terminate or disrupt translation from theRNA.

Non-ionic oligonucleotide analogs, i.e., oligomers with unchargedbackbones, generally cross cell membranes more readily than theircharged counterparts. Non-ionic oligonucleotide analogs includephosphotriester- and methylphosphonate-linked DNA (Miller et al.,Biochemistry 18:5134 (1979); Miller et al., J. Biol. Chem. 255:6959(1980)), carbamate-linked nucleosides (Stirchak, E. P. et al., J. Org.Chem. 52:4202 (1987)), phosphoroamidate-linked DNA (Froehler et al.,Nucleic Acids Res. 16:4831 (1988)), and peptide nucleic acids (PNAs).

A preferred nonionic antisense oligomer for use in the method of theinvention is an uncharged-backbone morpholino oligomer as defined above.Morpholino oligomers, such as illustrated in FIGS. 5 and 6, are composedof morpholino subunit structures linked together by uncharged,phosphorous-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit. Linked to each subunit is a purine or pyrimidinebase-pairing moiety effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The pyrimidine base-pairingmoieties may also include a C-5-propyne modification of thymidine and/orcytidine moieties, and/or a C-5-methyl modification of cytidine bases.

FIGS. 6AA-EE illustrate preferred backbone structures, showing twomorpholino subunits of a multisubunit oligomer. Each ring structureincludes a purine or pyrimidine or related hydrogen-bonding moiety,represented by P_(i) and P_(j), attached to the backbone morpholinemoiety through a linkage in the β-orientation. The purine or pyrimidinebase-pairing moieties in the oligomer are typically adenine, cytosine,guanine, uracil or thymine. In the structures of FIG. 5, the atom Ylinking the 5′ morpholino carbon to the phosphorous group may be sulfur,nitrogen, carbon, or oxygen; nitrogen and oxygen are preferred, andoxygen is particularly preferred. Z represents sulfur or oxygen, and ispreferably oxygen. The X moiety pendant from the phosphorous may be anyof the following: fluorine, alkyl or substituted alkyl, alkoxy orsubstituted alkoxy, thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures. Several cyclic disubstituted nitrogen moieties whichare suitable for the X moiety are morpholine, pyrrole, and pyrazole.Preferred embodiments of X are alkoxy, amino (NH₂) anddialkyl-substituted nitrogen.

In preferred embodiments of FIGS. 6B-B, Z is oxygen, Y is oxygen, and Xis alkoxy (phosphoramidate linkage), or Z is oxygen, Y is oxygen, and Xis unsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures (phosphorodiamidate linkage). Also preferred arelinkages in which Z is oxygen, Y is unsubstituted, monosubstituted, ordisubstituted nitrogen, and X is alkoxy (alternate phosphorodiamidatelinkage).

The solubility of the antisense compound, and the ability of thecompound to resist precipitation on storage in solution, can be furtherenhanced by derivatizing the oligomer with a solubilizing moiety, suchas a hydrophilic oligomer, or a charged moiety, such as a charged aminoacid or organic acid. The moiety may be any biocompatible hydrophilic orcharged moiety that can be coupled to the antisense compound and thatdoes not interfere with compound binding to the target sequence. Themoiety can be chemically attached to the antisense compound, e.g., atits 5′ end, by well-known derivatization methods. One preferred moietyis a defined length oligo ethylene glycol moiety, such as triethyleneglycol, coupled covalently to the 5′ end of the antisense compoundthrough a carbonate linkage, via a piperazine linking group forming acarbamate linkage with triethylene glycol, where the second piperazinenitrogen is coupled to the 5′-end phosphorodiamidate linkage of theantisense. Alternatively, or in addition, the compound may be designedto include one a small number of charged backbone linkages, such as aphosphodiester linkage, preferably near one of the ends of the compound.The added moiety is preferably effective to enhance solubility of thecompound to at least about 30 mgs/ml, preferably at least 50 mgs/ml inaqueous medium. Antisense oligomers of the present invention can alsoinclude modifications including, but not limited to, conjugated moietiessuch as cholesterol; diamine compounds with varying numbers of carbonresidues between the amino groups; and terminal ribose, deoxyribose andphosphate modifications which cleave or crosslink to hybridized targetnucleic acids or to associated enzymes or other proteins which bind tothe target nucleic acids.

Morpholino oligomers afford high target binding affinity, especially forRNA targets, and are resistant to degradation by nucleases. Binding of amorpholino oligomer to a target has been shown to give stronginactivation, due to the greater binding affinity noted above, andbecause the oligomer/target duplex is not susceptible to duplexunwinding mechanisms in the cell. Further, in therapeutic applicationsinvolving cellular uptake of the compound, the uncharged morpholinopolymer is more efficiently transported into cells than are oligomerswith charged backbones.

Although targeting of a messenger RNA sequence or an unspliced pre-mRNAsequence is preferred, a double-stranded DNA, i.e., a genomic DNA, maybe targeted by using a non-ionic probe designed for sequence-specificbinding to major-groove sites in duplex DNA. Oligomers suitable forforming base-specific triplex structures with a target duplex DNA aredescribed, for example, in co-owned U.S. Pat. No. 5,405,938.

In vivo Effectiveness of Moipholino Oligomers

Morpholino oligonucleotides have been shown to provide significantlyimproved selectivity in inhibiting translation of targeted sequences incomparison to phosphorothioate oligonucleotides, which are widely usedin the field. The morpholino oligomers have also been shown to inhibittranslation at much lower concentrations than the correspondingphosphorothioates, and with little or no evidence of the substantialnon-antisense activity exhibited by the phosphorothioates. See, forexample, Summerton et al., Antisense & Nucleic Acid Drug Dev 7 (2)p63-70 (1997). Because the morpholino oligomers are uncharged, they aremore effective at penetrating cell membranes. The morpholino oligomerswere also reported to have very high nuclease resistance and good watersolubility, making them good candidates for in vivo use.

The efficacy of morpholino antisense oligonucleotides in vivo isdescribed in co-owned and copending U.S. provisional application serialNo. 60/117,846, filed Jan. 29, 1999. In the procedures describedtherein, a phosphoramidate morpholino oligonucleotide (PMO) forms aheteroduplex with target RNA, which is protected in this duplex statefrom nuclease degradation. The duplex is expelled from the cell, and thetarget RNA can later be detected in a body fluid sample, e.g. a urinesample, from the subject. These results demonstrate that the morpholinooligomers (i) migrate to and enter cells in the body and (ii) bind withhigh affinity, via Watson-Crick base-pairing, to target nucleic acidregions, (iii) be expelled from the cells into the bloodstream in theform of a nuclease-resistant heteroduplex, and (iv) survive in thebloodstream in sufficient amount for detection in a body fluid.

III. Selection of Target Genes

The present invention relates to a method of improving thepharmacokinetics of a drug administered to a subject, by reducing theproduction of a drug-metabolizing enzyme which is up-regulated either bythe drug itself or by a xenobiotic agent to which the subject has beenexposed. The drug-metabolizing enzyme may convert the drug into a toxicmetabolite, reduce the half-life of the drug in the subject, or both.Transcription of target RNA from the enzyme gene (i.e., the “targetgene”) is induced by the drug, or by a different xenobiotic agent.According to the invention, a nuclease-resistant antisense oligomer,preferably a morpholino oligomer, is targeted to a gene encoding adrug-metabolizing enzyme which reduces the half-life of the drug, orconverts the drug into a toxic metabolite, or both. The antisenseoligomer, preferably co-administered with the drug, is effective toreduce production of the enzyme in the subject by hybridizing to thetarget RNA.

The production of any drug-metabolizing enzyme encoded by an endogenousxenobiotic-inducible gene may be decreased by the method of thisinvention. For example, important Phase 2 drug-metabolizing enzymesinclude epoxide hydrolases, whose substrates includecarbamazepine-10,11-epoxide; glucuronyl transferases, whose substratesinclude oxazepam, and which are induced by anticonvulsant drugs such asphenytoin and carbamazepine; and glutathione transferases. Selection ofthe enzyme will be determined by the metabolic scheme of the drug inquestion.

A. The Cytochrome p450 Family

In accordance with a preferred embodiment, the present inventionprovides antisense oligomers which are antisense to cytochrome p450(CYp450) genes. The cytochrome p450s are a collection of enzymesinvolved in the oxidative metabolism of both endogenous and exogenouscompounds. Over 200 genes encoding cytochrome p450, divided among over30 gene families, have been identified. The p450 gene families areorganized into subfamilies, which vary in regulation of gene expressionand in amino acid sequence homology, substrate specificity, catalyticactivity, and physiological role of the encoded enzymes. The followingdiscussion of representative p450 genes, inducers of those genes, andsubstrates of the encoded enzymes, is provided for illustrative purposesand is not intended to limit the invention.

Sequences for numerous p450 genes of various species are known andavailable to those of skill in the art through public databases such asGenBank and review articles such as F. J. Gonzales, “The MolecularBiology of Cytochrome p450's”, Pharmacological Reviews 40(4), 243-288(1989); S. D. Black et al., “P-450 Cytochromes: Structure and Function,”Adv. Enzymol. Relat. Areas Mol. Biol. 60, 35-87 (1987); D. R. Nelson etal., “The p450 Superfamily: Update on New Sequences, Gene Mapping,Accession Numbers, Early Trivial Names of Enzymes, and Nomenclature,”DNA Cell Biol. 12(1),1-51 (January-Febuary 1993), and articles citedtherein.

B. Sequence Homology of p450 Enzymes and Genes

High levels of sequence homology have been found among p450 enzymes andtheir genes in different mammalian species. For example, the rat, humanand rabbit 2E1 cDNAs have been isolated and sequenced, and their aminoacid sequences exhibit about 8.0% similarity. The human 2B subfamilycDNAs were isolated by screening liver libraries with rat 2B cDNAprobes, and the isolated cDNAs demonstrated >75% amino acid similarity.The rat CYP3A enzymes, CYP3A1 and CYP3A2, are approximately 90%identical and functionally equivalent to human CYPs 3A3 and 3A4,respectively (Desjardins and Iversen, J. Pharmacol. Exp. Ther.275(3):1608-13, 1995). The following table shows sequence similaritiesamong the ATG regions of mRNA from CYP3A genes of these and otherspecies.

TABLE 1 p450 GenBank SEQ ID Species Gene Accession No Sequence (5′ → 3′)NO: Rat CYP3A2 U09742 GAC AGA CAA GCA GGG ATG GAC CTG CTT TCA GCT 1X62087 S45634 Mouse CYP3A16 D26137 GAC AGA CAA GCA GAG ATG AAC CTA TTTTCA GCG 2 Mouse CYP3A16 X63023 TTA AAG AAA ACA GCA ATG GAC CTG ATC CCAAAC 3 Mouse CYP3AM1 X60452 GAC AAA CAA GCA GGG ATG GAC CTG GTT TTC AGC 4Hamster CYP3A D16363 AAA TCG CAC AAG GAA ATG GAC CTG GTC CCC AGC 5Rabbit CYP3A6 J05034 AGA AGG ACA GTG GCG ATG GAT CTG ATC TTT TCC 6 DogCYP3A12 X54915 AGA GGA CGA GTG GTC ATG GAC TTC ATC CCA AGC 7 Pig CYP3A39Z93099 ACG AGG ACA GTG GCC ATG GAC CTG ATC CCA GGC 8 Goat CYP3A24 U59378GCC AAG AAA GTG GCC ATG GAG CTG ATC CCA AGT 9 Monkey CYP3A S53047 GGAAGG AAA GTA GTG ATG GAT CTC ATC CCA GAC 10 Human CYP3A3 X12387 GTA AGGAAA GTA GTG ATG GCT CTC ATC CCA GAC 11 M13785 D00003 Human CYP3A4 M14096GTA AGG AAA GTA GTG ATG GCT CTC ATC CCA GAC 12 Human CYP3A5 J04813 AGAAGG AAA GTG GCG ATG GAC CTC ATC CCA AAT 13 Human CYP3A5A L35912 AGA AGGCAA GTG GCG ATG GAC CTC ATC CCA AAT 14 Human CYP3A7 D00408                GTG ATG GAT CTC ATC CCA AAC 15

The following degrees of homology were found in three conserved domainsof the p4503A proteins:

1. Heme-binding cysteine-containing peptide; the fifth ligand to hemeiron (23 amino acid segment starting at position 435 in human protein)Dog 21/23 91.3% similarity to human protein Rat 23/23 100% Mouse 22/2395.7% Monkey 21/23 91.3% Pig 22/23 95.7% Rabbit 20/23 87.0% 2. Membranetransition binding domain (8 amino acid segment starting at position 39in human protein) Dog 8/8 100% similarity to human protein Rat 7/8 87.5%Mouse 7/8 87.5% Monkey 7/8 87.5% Pig 7/8 87.5% Rabbit 8/8 100% 3. Signalsequence and half-transfer sequence for membrane insertion (20 aminoacid segment starting at position 167 in human protein) Dog 17/20 85%similarity to human protein Rat 18/20 90% Mouse 18/20 90% Monkey 20/20100% Pig 19/20 95% Rabbit 16/20 80% Goat 16/20 80% Guinea pig 13/20 65%The average degree of homology over these three known functional domains(51 amino acids of 500 total) was 92.9% human to monkey, 92.5% human torat, and 92.1% human to dog.

C. Substrates and Inducers of p450 Enzymes

Genes in the CYP2B subfamily are known to be strongly induced byphenobarbital. The 2B1 and 2B2 proteins exhibit 97% amino acidsimilarity. These enzymes have similar substrate specificities; however,purified 2B1 (rat) has about a 5-fold higher catalytic activity than 2B2for certain substrates, including benzphetamine and testosterone, and atwo-fold to three-fold higher activity for the substrates benzo[a]pyreneand 7,12-dimethylbenzanthracene.

A distinct ethanol-inducible form of p450, CYP2E1, was first identifiedin rabbits and later in rats and humans. The enzymes of this subfamilymetabolize a large number of substrates, as shown below, including, forexample, ethanol, acetone, acetoacetate, acetol, diethyl ether,p-nitrophenol, halothane, benzene, pyridine, and N-nitrosodimethylamine.

The CYP3A subfamily is involved in the 6β-hydroxylation of testosteroneand in the metabolism of numerous clinically important drugs, such asthose listed below.

Listed below are further examples of known inducers and substrates ofmembers of various p450 subfamilies. See also the discussion in Klassen,ed., Casarett and Doull's Toxicology: The Basis Science of Poisons,McGraw-Hill, 1996, pp. 150 ff. Further information about cytochrome p450substrates, inducers, and metabolites can be found in Gonzales and otherreview articles cited above. Current information sources available viathe Internet include the “Cytochrome p450 Homepage”, maintained by DavidNelson, the “Cytochrome p450 Database”, provided by the Institute ofBiomedical Chemistry & Center for Molecular Design, and the “Directoryof p450-containing Systems”, provided by Kirill N. Degtyarenko and PéterFábián.

The exemplary p450 genes discussed herein are given for illustrativepurposes only and are not intended to limit the invention.

p450 Family 1 (CYP1)

CYP1A1:

inducers include: dioxin, PAR (polycyclic aromatic hydrocarbons) intobacco smoke or charcoal-broiled beef, β-naphthoflavone in food

substrates include: diethylstilbestrol, 2- and 4-hydroxyestradiol

CYP1A2:

inducers include: dioxin, PAH, β-naphthoflavone, cruciferous vegetables,omeprazole

substrates include: acetaminophen, phenacetin, acetanilide (analgesics),caffeine, clozapine (sedative), cyclobenzaprine (muscle relaxant),estradiol, imipramine (antidepressant), mexillitene (antiarrhythmic),naproxen (analgesic), riluzole, tacrine, theophylline (cardiacstimulant, bronchodilator, smooth muscle relaxant), warfarin.

probe reaction: caffeine 3-demethylation

p450 Family 2 (CYP2)

CYP2A6:

inducers include: barbiturates

substrates include: coumarin, butadiene, nicotine

CYP2B1:

inducers include: phenobarbital

substrates include: phenobarbital, hexobarbital

CYP2C9:

inducers include: rifampin, secobarbital

substrates include: NSAIDs such as diclofenac, ibuprofen, and piroxicam;oral hypoglycemic agents such as tolbutamide and glipizide;angiotensin-2 blockers such as irbesartan, losartan, and valsartan;naproxen (analgesic); phenytoin (anticonvulsant, antiepileptic);sulfamethoxazole, tamoxifen (antineoplastic); torsemide; warfarin

CYP2C19:

inducers include: rifampin, secobarbital

substrates include: hexobarbital, mephobarbital, imipramine,clomipramine, citalopram, cycloguanil, the anti-epileptics phenytoin anddiazepam, S-mephenytoin, diphenylhydantoin, lansoprazole, pantoprazole,omeprazole, pentamidine, propranolol, cyclophosphamide, progesterone

CYP2D6:

inducers include: dexamethasone, rifampin

substrates include: antidepressants (imipramine, clomipramine,desimpramine), antipsychotics (haloperidol, perphenazine, risperidone,thioridazine), beta blockers (carvedilol, S-metoprolol, propafenone,timolol), amphetamine, codeine, dextromethorphan, fluoxetine,S-mexilletine, phenacetin, propranolol

CYP2E1:

inducers include: ethanol, acetone, isoniazid, dimethyl sulfoxide,pyrazoles.

substrates include: acetaminophen; chlorzoxazone (muscle relaxant),ethanol; caffiene, theophylline; dapsone, general anesthetics such asenflurane, halothane, and methoxyflurane; nitrosamines

p450 Family 3 (CYP3)

CYP3A1, CYP3A2: rat CYP3A subfamily; approximately 90% identical andfunctionally equivalent to human CYP3A3 and CYP3A4, respectively (below)

CYP3A4:

inducers include: carbamazepine, phenobarbital, phenytoin, dexamethasoneand other glucocorticoids; barbiturates, various steroids, antibioticssuch as rifampin, rifabutin, erythromycin; phenylbutazone,sulfadimidine, sulfinpyrazone, troleandomycin

substrates include: HIV Protease Inhibitors such as indinavir,ritonavir, and saquinavir; benzodiazepines such as alprazolam, diazepam,midazolam, and triazolam; immune modulators such as cyclosporine;antihistamines such as astemizole and chlorpheniramine; HMG CoAReductase inhibitors such as atorvastatin, cerivastatin, lovastatin, andsimvastatin; channel blockers such as diltiazem, felodipine, nifedipine,nisoldipine, nitrendipine, and verapamil; antibiotics such asclarithromycin, erythromycin, and rapamycin; various steroids includingcortisol, testosterone, progesterone, estradiol, ethinylestradiol,hydrocortisone, prednisone, and prednisolone; acetominophen, aldrin,alfentanil, amiodarone, astemizole, benzphetamine, budesonide,carbemazepine, cyclophosphamide, ifosphamide, dapsone, digitoxin,quinidine (anti-arrhythmic), etoposide, flutamide, imipramine,lansoprazole, lidocaine, losartan, omeprazole, retinoic acid, FK506(tacrolimus), tamoxifen, taxol, teniposide, terfenadine, buspirone,haloperidol (antipsychotic), methadone, sildenafil, trazodone,theophylline, toremifine, troleandomycin, warfarin, zatosetron,zonisamide.

p450 Family 6 (CYP6)

CYP6A1:

inducers include: chlofibrate

substrates include: fatty acids

D. Exemplary Drugs Metabolized by p450 Enzymes

Acetaminophen

Ethanol up-regulates CYP2E1, which metabolizes acetaminophen to areactive quinone (FIG. 2). This reactive quinone intermediate, whenproduced in sufficient amounts, causes liver damage and necrosis. Anoligomer antisense to the CYP2E1 gene reduces synthesis of the enzymeand decreases production of the toxic intermediate. Reducing the fluxthrough the toxic pathway enables alternative, more desirable metabolicpathways to compensate.

Sedatives

The sedative phenobarbital (PB) up-regulates several p450 genes,including those of the CYP2B and CYP3A subfamilies. Upregulation ofthese enzymes increases the metabolism and reduces the sedative effectsof PB and the related sedative hexobarbital. Example 1 demonstrates thatan antisense oligonucleotide to the CYP2B1 gene reduces synthesis of theenzyme and decreases HB metabolism, enabling lower amounts of HB beadministered for the equivalent sedative effect. Such oligomers alsoincreased the effectiveness of HB in the presence of the inducing agentphenobarbital. Example 3 demonstrates a similar effect of antisense toCYP3A2 on efficacy of midazolam (MZ).

Antibiotics

The antibiotics rifampicin, rifampin, rifabutin, erythromycin, andrelated compounds are inducers of the CYP3A4 gene and are substrates ofthe enzyme product. An oligomer antisense to the CYP3A4 gene increasesthe serum half-life and hence the effectiveness of the antibiotic.

Oral Contraceptive/estrogen Replacement Therapy

Estrogens and estradiols are the active ingredients in oralcontraceptives and in hormonal replacement therapies for post-menopausalwomen. Women who are also taking antibiotics such as rifampicin orerythromycin, or glucocorticoids such as dexamethasone, or who smoke,risk decreased efficacy of the estrogen/estradiol treatments due toincreased metabolism of these compounds by up-regulated CYP3A4 and/orCYP1A2 enzymes. Administration of oligomers antisense to the CYP3A4and/or CYP1A2 genes in such situations block up-regulation of theseenzymes and reduces risk of pregnancy in women taking oralcontraceptives, or of osteoporosis in women receiving estrogenreplacement therapy.

Protease Inhibitors

All protease inhibitors and non-nucleoside reverse transcriptaseinhibitors currently indicated for use in treatment of HIV aresubstrates of p450 enzymes; in particular, they are metabolized byCYP3A4 enzymes (see e.g. Sahai, AIDS 10 Suppl 1:S21-5, 1996) withpossible participation by CYP2D6 enzymes (Kumar et al., J. Pharmacol.Exp. Ther. 277(1):423-31, 1996). Although protease inhibitors arereported to be inhibitors of CYP3A4, some non-nucleoside reversetranscriptase inhibitors, such as nevirapine and efavirenz, are inducersof CYP3A4 (see e.g. Murphy et al., Expert Opin Invest Drugs 5/9:1183-99, 1996). Given the increasing use of multidrug therapy fortreatment of HIV infection, the potential for interference is high.Supplemental administration of oligomners antisense to CYP3A4 and/orCYP2D6 genes can block up-regulation of these enzymes, thus reducing themetabolism of the protease inhibitors, allowing for lower doses andreduction of sometimes serious side effects.

IV. Design and Preparation of Antisense Oligomers

A. Selection of Target Sequences

Target sequences, including genomic sequences, pre-mRNA, mRNA, and/orcDNA sequences, from genes selected according to the considerationsoutlined in the previous sections, may be obtained from the GenBanksequence database or from other published sources readily available tothose of skill in the art. As noted above, sequences for numerous ratand human p450 genes are known and available to those of skill in theart through sources such as GenBank and review articles such as Gonzales1989, Black et al. 1987, and Nelson et al. 1993, cited above. Forexample, Nelson et al. lists all database accession numbers for p450genes that were available in the GenBank/EMBL, SwissProt, and NBRF-PIRdatabases as of December 1992. Accession numbers for human p450sequences are included from the following families: CYP-1A1, 1A2, 2A6,2A7, 2B6, 2B7P, 2C8, 2C9, 2C10, 2C17, 2C18, 2C19, 2D6, 2D7P, 2D8P, 2E1,2F1 3A3, 3A4, 3A5, 3A7, 4A9, 4A11, 4B1, 4F2, 4F3, 5, 7, 11A1, 11B1, 17,19, 21A1P, 21A2, and 27. Since the publication of the 1993 article,other human sequences, such as those for CYP-1B1 and CYP-2B1, have alsobeen made available in GenBank.

B. Length and Complementarity of the Antisense Oligomer

The appropriate length of the antisense oligomer to allow stable,effective binding combined with good specificity is about generally 10to 40 nucleotide base units, and preferably about 15 to 25 base units.The antisense oligomer may be 100% complementary to a desired region ofthe target sequence, or it may include mismatches, e.g., to accommodatecoding sequence variants, such as polymorphisms, as long as the duplexformed between the oligomer and target RNA is sufficiently stable in thecell to block or inhibit translation. Mismatches, if present, are lessdestabilizing toward the end regions of the hybrid duplex than in themiddle. The number of mismatches allowed will depend on the length ofthe oligomer, the percentage of G:C basepair in the duplex and theposition of the mismatch(es) in the duplex, according to well understoodprinciples of duplex stability. Preferably, the T_(m) of theoligomer/target sequence will be at least 37° C., more preferably atleast 50° C., and most preferably at least 60° C., 80° C., or higher.Oligomer bases that allow degenerate base pairing with target bases arealso contemplated, assuming base-pair specificity with the target ismaintained.

C. Exemplary Antisense Oligomers Targeting p450

Exemplary oligomer sequences can be designed according to the followingguidelines:

1. Each oligomer either (a) spans the AUG start codon of the indicatedgene, with the CAU complement of the start codon (expressed in a 5′ to3′ direction) being positioned near the center or near the 3′ end of theoligomer, or (b) spans an intron-exon (splice donor) boundary or, morepreferably, an exon-intron (splice acceptor) boundary of an unsplicedpre-mRNA sequence;

2. has a length of about 20-25 bases; and

3. preferably terminates, at the 5′ end, at a G (guanine) base, whichconfers stability to the duplex.

Exemplary antisense oligomers having the base sequences shown in Table 2are designed for p450 RNA-specific inhibition of translation and/orsplicing. The location of the bases in the target sequence whichhybridize with the oligomer, numbered according to the GenBank sequencenumbering, is indicated at the right in the table. By convention, theorientation of the antisense sequences is shown in a 5′ to 3′ direction.In a hybrid duplex in which the target gene sequence is shown a 5′ to 3′direction (by convention), the orientation of the hybridized antisenseoligomer sequence would be reversed; that is in a 3′ to 5′ direction.The table also identifies the sequence identifier number (SEQ ID NO:) ofeach exemplary oligomer sequence. Preferred antisense oligomers for usein practicing the method of the invention are those identified by SEQ IDNOs: 16-17, 19, 25, 27, 30, 34-35 and 46-47, for inhibiting translationand/or splicing of target RNAs derived from the rat CYP2B1, CYP2E1, andCYP3A2 and human CYP1A2, CYP2B1, CYP2E1, and CYP3A4 genes, respectively.

TABLE 2 SEQ p450 GenBank Antisense Sequence ID Site Posn. in Gene Acc.No. (5′ → 3′) NO Targeted sequence CYP2B1 M11251 GGAGCAAGATACTGGGCTCCAT16 ATG start 490- (rat) J00719 AAAGAAGAGAGAGAGCAGGGAG 17 downstream 855-of ATG CYP2E1 M20131 GGTTTATTATTAGCTGCAGTTGGCTATCAT 18 upstream of 1406-ATG CCAAGAACCGCCATGGTGCC 19 ATG start 1560- ACCTTGGTGAAAGACTTGGG 20 exon1 1725- splice donor CCTTGTTCTTGTACTCCTGG 21 exon 2 2645- splice donorGAGAAGCATGGTCACCTGGA 22 exon 4 6681- splice donor CCAACACACACACGCTTTCC23 exon 9 11591- splice acceptor CYP3A2 U09742 TGAGAGCTGAAAGCAGGTCCAT 24ATG start 69- GAGCTGAAAGCAGGTCCATCCC 25 " 66- CYP1A1 K03191ATTGGGAAAAGCATGATCAG 26 ATG start 81- (human) CYP1A2 L00384TGGGACAATGCCATCTGTAC 27 " 9- M14337 CYP1B1 U03688 AGGCTGGTGCCCATGCTGCG28 " 341- CYP2A6 M33318 CCTGAGGCCAGCATGGTGGT 29 " 4- M33316 CYP2B1M29874 ACGCTGAGTTCCATGGTCTG 30 " 1- J02864 CYP2C9 M61855ACAAGAGAATCCATTGAAGC 31 " 7- J05326 CYP2C19 M61854 CACAAAAGGATCCATTGAAG32 " 1- J05326 CYP2D6 M33388 GCTTCTAGCCCCATACCTGC 33 " 1614- CYP2E1J02843 CCGAGGGCAGACATGGTGCC 34 " 2819- CYP3A4 AF182273GTCTGGGATGAGAGCCATCAC 35 " 7-   CTGGGATGAGAGCCATCAC 46 " 7-  CTGGGATGAGAGCCATCACT 47 " 6-

E. Testing the Effectiveness of the Antisense Oligomers

The effectiveness of a given antisense oligomer molecule in inhibitingexpression of the target gene may be determined by screening methodsknown in the art.

E1. In vitro Screening Methods

Candidate antisense oligomers can be tested in vitro in, for example,hepatocyte cell culture, to quantify the effect of the oligomer onprotein produced by the target RNA in the presence and absence of drugsor other inducers. See, for example, Examples 2 and 4 below. RNAquantitation methods are known in the art, and include Northern blot andRT-PCR assays. Protein production can be evaluated by Western blot orELISA using antibodies specific for the target enzyme. Alternatively orin addition, protein expression can be evaluated enzymatically usingprobe substrates specific for the target enzyme. For example, substratesare known which differentially react with various p450 isozymes (M.Burke et al., Biochem. Pharmacol. 34(18):3337-45 (1985); Gonzalez, 1989(cited above)).

Candidate oligomers are also evaluated, according to well known methods,for acute and chronic cellular toxicity, such as the effect on overallprotein and DNA synthesis as measured via incorporation of ³H-leucineand ³H-thymidine, respectively.

It is desirable that non-specific binding of the oligomeric molecule tonon-target sequences is limited. To test for non-specific bindingeffects, control sequences such as sense or nonsense sequences, orsequences containing mismatched bases, may be included in screeningtests. Excess targeted protein or mRNA may also be added to the cellculture to determine if the effect of the antisense oligomer isreversed.

E2. In vivo Screening Methods

Antisense compositions may be tested in vivo in animal models asdescribed, for example, in Examples 1, 3, and 5 below. Effects of a drugcan be observed directly, as in sleep time induced by hexobarbital,and/or enzyme levels can be determined by assays known in the art.

As described above and in copending and co-owned U.S. application serialNo. 60/117,846, duplexes of PMO antisense oligomers with target RNAsequences have been detected in body fluid samples following in vivoadministration of the PMO oligomers. Such methods could be employed forin vivo screening of target RNA binding of a given oligonucleotide.

V. Pharmacokinetics and Administration

The pharmacokinetics of nuclease-resistant oligonucleotides has beenshown to be favorable for in vivo therapeutic treatment of variousendogenous genes. To date, studies in the rat, mouse and monkey revealan elimination half-life in plasma longer than twenty hours. It has alsobeen shown that the liver, where most drug metabolism occurs, is anorgan of accumulation for oligonucleotides. See, for example, P. Iversenet al., Antisense Res & Dev 4: 43-52 (1994) and E. Bayever et al.,Antisense Res and Dev 3:383-390 (1993), for discussions of thepharmacokinetics of phosphorothioate oligonucleotides administered toanimal subjects and to human patients. Bayever et al. conclude thatsafety and favorable pharmacokinetics support further investigations ofphosphorothioate oligonucleotides as potential gene specific therapeuticagents in humans.

In a preferred embodiment of the present method, the antisenseoligonucleotide is a morpholino oligonucleotide, particularly aphosphoramidate- or phosphorodiamidate-linked morpholino oligonucleotide(PMO). These molecules have been shown to provide significantly improvedselectivity in inhibiting translation of target sequences in comparisonto the widely usephosphorothioates. The morpholino oligomers were alsoshown to inhibit translation at much lower concentrations than thecorresponding phosphorothioates, and with little or no evidence of thenon-antisense activity often exhibited by phosphorothioates. See, forexample, Summerton et al., Antisense & Nucleic Acid Drug Dev 7(2):63-70(April 1997). Because the morpholino oligomers are uncharged, they aremore effective at penetrating cell membranes. The morpholino oligomersalso have high nuclease resistance and good water solubility, makingthem good candidates for in vivo use.

Table 3 compares pharmacokinetic and renal excretion properties ofphosphorothioate (PS) and PMO oligonucleotides, such as those shown inTable 8 below, after IP injection of a single 0.1 mg dose.

TABLE 3 Single Dose (0.1 mg) Plasma Pharmacokinetics PS PMOPharmacokinetic Properties Half-life 7.8 ± 3.8 hrs 7.1 ± 1.9 hrs Volumeof 1.2 ± 0.3 liters/kg 1.5 ± 0.2 liters/kg Distribution Area Under Curve245.4 ± 13.3 μg · min/ml 337 ± 67 μg · min/ml Plasma Clearance 0.43 ±0.02 ml/min 1.2 ± 0.3 ml/min Renal Excretion Properties Excretion Rate4.8 ± 0.6 ng/min 15.9 ± 3.1 ng/min Renal Clearance 3.4 ± 1.5 ml/min 0.75± 0.18 ml/min

Routes of administration of antisense oligomers include, but are notlimited to, various systemic routes, including oral and parenteralroutes, e.g., intravenous, subcutaneous, intraperitoneal, intramuscular,and intraarterial injection, as well as inhalation and transdermaldelivery. In some cases targeted delivery by direct administration to aparticular tissue or site is preferred. It is appreciated that anymethods which are effective to deliver the drug to a target site or tointroduce the drug into the bloodstream are also contemplated. In apreferred embodiment, the oligomer is a morpholino oligomer, iscontained in a pharmaceutically acceptable carrier, and is deliveredorally.

Examples of standard pharmaceutically accepted carriers include saline,phosphate buffered saline (PBS), water, aqueous ethanol, emulsions suchas oil/water emulsions, triglyceride emulsions, wetting agents, tabletsand capsules. It will be understood that the choice of suitablephysiologically acceptable carrier will vary dependent upon the chosenmode of administration. For example, transdermal delivery of antisenseoligomers may be accomplished by use of a pharmaceutically acceptablecarrier adapted for topical administration.

Molecular conjugates useful for delivering antisense morpholinooligomers are described in U.S. Pat. No. 6,030,941 (Summerton andWeller, 2000), which is incorporated herein by reference. The oligomersof the invention may also be administered encapsulated in liposomes.(See, e.g., Williams, S. A., Leukemia 10(12):1980-1989, 1996;Lappalainen et al., Antiviral Res. 23:119, 1994; Uhlmann et al.,“Antisense Oligonucleotides: A New Therapeutic Principle”, in ChemicalReviews, Volume 90, No. 4, pp 544-584, 1990; Gregoriadis, G., Chapter14, “Liposomes”, in Drug Carriers in Biology and Medicine, pp 287-341,Academic Press, 1979.) The active ingredient, depending upon itssolubility, may be present both in the aqueous phase and in the lipidiclayer(s), or in what is generally termed a liposomic suspension. Thelipidic layer generally comprises phospholipids, such as lecithin orsphingomyelin, steroids such as cholesterol, ionic surfactants such asdiacetylphosphate, stearylamine, or phosphatidic acid, and/or otherhydrophobic materials. The diameters of the liposomes generally rangefrom about 15 nm to about 5 microns.

In practicing the method of the invention, the antisense oligomer isco-administered with the drug at a desired dose and dosing schedule.Preferably, the oligonucleotide is first administered several hours toseveral days before first administering the drug, to allow reduction ofthe target enzyme level. Preferred doses for oral administration arebetween about 1-2 mg oligomer/kg patient body weight, assuming anoligonucleotide MW of about 7000. A typical therapeutic dose for apatient weight 70 kg would thus be about 70 mg administered once a day,although higher doses may be administered if needed. For IVadministration, the preferred doses are about ⅓ the oral dose.

The dose may be administered once several times daily, once daily, orless often, e.g., for prophylactic purposes. The efficacy of treatmentmay be followed by established tests, according to the drug whosemetabolism is being modulated. Typically, the oligonucleotide will beadministered at least once daily for a period of time concurrent withthe administration of the drug, and may be discontinued, for example,when the drug therapy is discontinued.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Assay reagents were purchased from GenTest Corporation (Woburn, Mass.),which produces cytochrome p450 reagents and antibodies.

Example 1

Coadministration of Antisense to Rat Cytochrome p50 (CYP) 2B1 withHexobarbital

1A. Coadministration of Antisense Olijomers, Hexobarbital and/orInducing Agent Phenobarbital

Antisense oligonucleotides were designed to be complementary to targetsequences within the cytochrome p450 2B1 (rat CYP2B1) gene sequence,with the objective of improving the metabolism of hexobarbital in rats.Five phosphorothioate oligomers were synthesized according to the knownrat CYP2B1 sequence (GenBank Accession No. J00719).

The antisense oligomers are shown in Table 4. The oligo 2B1-ATG (SEQ IDNO: 16) is a 22-mer having 100% complementarity to a region containingthe AUG start codon on the rat CYP2B1 mRNA (SEQ ID NO:39, shown belowTable). The oligo 2B1-NRM (SEQ ID NO: 17) has 100% complementarity tothe CYP2B1 mRNA starting at base 855. The oligo 2B1-BPB (SEQ ID NO: 36)has the sequence of 2B1-NRM with a two base deletion (indicated in boldin the 2B1-NRM sequence). Each of the oligos 2B1-3MM (SEQ ID NO: 37) and2B1-CMM (SEQ ID NO: 38) has 2 bases reversed (indicated in bold)compared to the BPB sequence. These last three oligos were compared toall sequences in GenBank and did not show homology to any sequencelisted in the database. The table gives the melting temperature (T_(m)),molecular weight, and % homology to CYP2B1 mRNA of each sequence.

TABLE 4 Characteristics of ODNs Used in Example 1 SEQ ID % Identity NO:Name T_(m)° C. Mol.Wt. with CYP2B1 sequence 16 2B1-ATG 68.0 6900 1005′-GGAGCAAGATACTGGGCTCCAT-3′ 17 2B1-NRM 50.6 7799 100 5′-AAAGAAGAGA GAGAGCAGGGAG-3′ 36 2B1-BPB 47.0 7086 90 6′-AAAGAAGAGAGAGCAGGGAG-3′ 372B1-3MM 49.8 7086 80 5′-AAAGAAGAGAGAGCAGGG GA -3′ 38 2B1-CMM 49.5 708680 5′-AAAGAAGAGA AG GCAGGGAG-3′

 Sequence targeted by 2B1-ATG: 3′-CCTCCTCGTTCTATGACCCGAGGTACCA-5′  (SEQID NO: 39)

Sequence targeted by 2B1-NRM: 3′-TCGTTTCTTCTCTCTCTCGTCCCTCTAG-5′  (SEQID NO: 40)

Male Sprague-Dawley rats (Sasco, Omaha) weighing between 210 to 290grams were used for all studies. They were housed in animal quarters atthe University of Nebraska Medical Center AAALAC-approved AnimalResource Facility. The animals were exposed to 12 hour light/dark cycleand fed Purina rat chow and tap water ad libitum.

Rats were injected intraperitoneally (i.p.) with the indicated ODNs (seeTable 3) once per day for 2 days, in an amount of 1 mg/kg body weightper injection. Control rats were injected with saline only. Rats in the“PB” groups were injected i.p. with phenobarbital (Mallinckrodt, St.Louis) at 80 mg/kg body weight per injection, once per day for 2 days.In the groups in which PB was co-administered with ODN, injections wereadministered simultaneously. Total volume of injections for all groupswas 0.1 ml/mg body weight. Animals in the “2B1-NRM+PB+PRETREATMENT”group also received a dose of 1 mg/kg of 2B1-NRM oligomer (SEQ ID NO:17) 24 hours prior to the first administration of 2B1-NRM plus PB.

Forty-eight hours following the first (non-pretreatment) injection ofODN and/or PB, the rats were injected i.p. with 100 mg/kg body weighthexobarbital (Sigma, St. Louis), in a total volume of 1 ml/kg bodyweight, prepared fresh daily. Sleep time, defined as the time periodfrom when the rat is placed on its back to when it regains its rightingreflex, was measured. Sleep times are given in Table 5 as the mean ofeach animal in the group±standard deviation.

Treatment of the various group is summarized as follows:

1. injected with saline only.

2. injected with 80 mg/kg PB per day for 2 days.

3. injected with 1 mg 2B1-NRM per day for 2 days.

4. injected simultaneously with 1 mg ODN 2B1-NRM and 80 mg/kg PB per dayfor 2 days.

5. same as 4 except injected with 1 mg 2B1-NRM one day before start of2B1-NRM+PB injections.

6. injected with 1 mg ODN 2B1-ATG per day for 2 days.

7. injected simultaneously with 1 mg ODN 2B1-ATG and 80 mg/kg PB per dayfor 2 days.

8. injected with 1 mg ODN 2B1-BPD per day for 2 days.

9. injected with 1 mg ODN 2B1-3MM per day for 2 days.

10. injected with 1 mg ODN 2B1-CMM per day for 2 days.

TABLE 5 Hexobarbital Sleep Times Percent SEQ ID NO: Sleep Time of NumberGroup of oligomer (min ± sd) Control Observed 1. Control — 23.4 ± 4.0100.0 10 2. Phenobarbital (PB) — 11.4 ± 4.5^(a) 48.7 5 3. 2B1-NRM 1723.0 ± 3.2 98.2 4 4. 2B1-NRM + PB 17 13.5 ± 0.9 57.7 4 5. 2B1-NRM 1713.3 ± 3.0 56.8 4 Pretreatment + PB 6. 2B1-ATG 16 20.5 ± 5.3 87.6 4 7.2B1-ATG + PB 16 19.3 ± 4.4^(b) 82.5 4 8. 2B1-BPD 36 8.6 ± 8.3^(c) 36.7 89. 2B1-3MM 37 31.0 ± 9.6 132.4 3 10. 2B1-CMM 38 23.0 ± 5.3 98.2 3^(a)Significantly different from control (p < .05). ^(b)Significantlydifferent from phenobarbital treated group (p < .03). ^(c)Significantlydifferent from control (p < .001), 2B1-3MM (p < .001) and 2B1-CMM (p <.001).

Control rats (injected only with saline during the 2-day treatmentregimen) had a sleep time of about 23 minutes. PB-treated rats showed asleep time of about 11.4 minutes, a significant reduction in sleep timeover control rats, as expected. As PB stimulates CYP2B1 gene expression,hexobarbital (which is hydroxylated by CYP2B1) was more quicklymetabolized in the PB-treated rat and its sedative effect was reduced.

When administered alone, oligomer 2B1-NRM, SEQ ID NO: 17 (Group 3) hadno effect on sleep time compared to control, which could be attributedto a low constitutive (i.e. non-induced) level of expression of CYP2B1in the rat.

Oligomer 2B1-NRM & PB administered together (Group 4) had no observableantisense effect. Since PB induction of gene expression is rapid (30-60minutes) (Waxman and Azaroff, Biochem J. 281(3):577-92,1992), and ODNaccumulation in the liver is slow (ca. 12 hours, as shown in relatedstudies by the inventor), rats in one group were pretreated with 2B1-NRMone day before simultaneous injection of PB+2B1-NRM (Group 5). However,the resulting sleep time was essentially the same as withoutpretreatment.

Oligomer 2B1-ATG (SEQ ID NO: 16) alone (Group 6) did not significantlyalter sleep time over control, again possibly due to low constitutiveexpression of CYP2B1. When 2B1-ATG and PB were injected simultaneously,however (Group 7), sleep time increased significantly over that of thePB group. Oligomer 2B1-ATG thus showed a potent in vivo antisense effecttoward PB-induced CYP2B1 expression.

Oligomer 2B1-3MM alone (SEQ ID NO: 37) slightly lengthened sleep timeover the control. Oligomer 2B1-BPD (SEQ ID NO: 36) significantly reducedsleep time from control, suggesting an anomalous increase in HBmetabolism. Oligomer 2B1-CMM (SEQ ID NO: 38) did not significantly alterHB sleep time from the control.

1B. Preparation of Microsomes

Microsomes were prepared, as described by Franklin and Estabrook (Arch.Biochem. Biophys. 143:318-29, 1971), for determination of enzyme leveland activity. The rats were sacrificed using ethyl ether, and liverswere perfused with 12 ml of 4% saline via the portal vein and thenremoved from the animal. The livers were minced, homogenized in 0.25 Msucrose (Sigma) and centrifuged at 8000×G for 20 minutes at 4° C. in aSorvall RC2-B centrifuge (Dupont). The supernatant was saved,resuspended in 0.25 M sucrose, and centrifuged at 100,000×G for 45minutes at 4° C. in a Sorvall OTD55B ultracentrifuge (Dupont). Thepellet was resuspended in 1.15% KCl (Sigma) and centrifuged at 100,000×Gfor 1 hour at 4° C. The final pellet was resuspended in an equal volumebuffer (10 mM Tris-acetate, 1 mM EDTA, 20% glycerol; Sigma) and frozenat −80° C.

1C. Determination of Protein Concentrations

Protein concentrations were determined by Bradford assay (M. M.Bradford, Anal Biochem 72:248-54, 1976). Eighty μl aliquots ofhomogenate, prepared as described above, were added to a 96 well plate(Becton Dickinson Labware, Lincoln Park, N.J.). Twenty μl of Bradfordreagent (Bio-Rad Richmond, Calif.) was then added and the plates read at595 nm on the microplate reader (Molecular Devices, Newport Minn.). Thedata was compared to standard curve generated with known concentrationsof bovine serum albumin (Sigma).

1D. Determination of Enzyme Activity: PROD Assay

CYP2B1 enzyme activity was determined by pentoxyresorufin O-dealkylation(PROD) activity (Burke et al., 1985). For each microsomal sample, 1 mgprotein in 1 ml 0.1 M potassium phosphate buffer, 1 ml 2 μM5-pentoxyresorufin (Pierce, Rockford, Ill.), and 17 μl 60 mM NADPH weremixed and incubated for 10 minutes at 37° C. The mixture was then addedto a 2 ml cuvette and read on a RF5000U spectrofluorophotometer(Shimadzu, Columbia, Md.), using an excitation wavelength of 530 nm andemission wavelength of 585 nm. Concentrations were calculated from acalibration curve of resorufin standards (Pierce, Rockford, Ill.).Results were recorded in nmol resorufin/mg protein/min.

Activity of microsomes from control rats was 13.8±10.1 nmol resorufin/mgprot/min. Microsomes isolated from PB groups all had significantlyincreased PROD activities, ranging from about 50 to 115 nmolresorufin/mg prot/min. Microsomes from 2B1-NRM, 2B1-ATG, 2B1-3MM, and2B1-CMM treated rats (SEQ ID NOs: 17, 16, 37 and 38, respectively)showed no change from control. Although not significantly different fromcontrol, 2B1-BPD (SEQ ID NO: 36) showed a small increase in activity.(In interpreting these results, it should be noted that thepentoxyresorufin O-dealkylation (PROD) assay measures both CYP2B1 andCYP2B2.)

1E. ELISA Assay

Direct measurement of CYP2B1 protein was performed by an ELISA assay,using an antibody directed to the CYP2B1 protein (Schuurs and VanWeeman, Clin Chim Acta 81(1):1-40, 1977). Fifty μg of microsomalproteins per well were plated in 100 μl 0.35% sodium bicarbonate bufferovernight on a 96 well nunc-immuno plate (InterMed, Skokie, Ill.). Themicrosomes were washed 3× with 1% bovine serum albumin in PBS (PBS/BSA)and incubated for 1 hr at 37° C. with 200 gl PBS/BSA. The PBS/BSA wasremoved, and 50 μl of CYP2B1 antibody (Oxygene, Dallas) was added andincubated for 1 hour at 37° C. The microsomes were washed 5× withsaline/Tween 20™ (Sigma), and 50 μl horseradish peroxidase(HRP)-conjugated secondary antibody (Bio-Rad) was added. The microsomeswere incubated for 1 hour at 37° C., then washed 5× with saline/Tween20™ and twice with 85% saline. HRP substrate (Kirkegaard & Perry Labs,Gaithersburg, Md.) (100 μl) was added, and the absorbance at 405 nm wasread continuously in a microplate reader (Molecular Devices) for 1 hour.Results were recorded as HRP activity in mOD/min.

The results are shown graphically in FIG. 3. The average HRP activitiesand n values for the various groups are given in Table 6.

TABLE 6 SEQ ID NO: Group of oligomer HRP activity n = Control — 14.2 ±3.0 8 PB alone — 22.5 ± 4.2 5 2B1-NRM 17 13.8 ± 5.6 4 2B1-NRM + PB 1717.9 ± 4.6 4 2B1-NRM + PB/Pretreatment 17 18.7 ± 0.3 4 2B1-ATG 16 13.2 ±4.4 4 2B1-ATG + PB 16 12.9 ± 5.0 5 2B1-BPD 36 25.1 ± 6.6 3 2B1-3MM 379.2 ± 5.0 3 2B1-CMM 38 10.1 ± 3.3 3

As expected, HRP activity of microsomes treated with PB alone wassignificantly greater than the activity of microsomes from control rats,due to induction of expression of the CYP2B1 gene by PB. Microsomes ofrats treated with oligomers 2B1-NRM, 2B1-ATG, 2B1-3MM, and 2B1-CMM (SEQID NOs: 17, 16, 37 and 38, respectively) showed no change in HRPactivity over control microsomes.

Microsomes from rats in the 2B1-NRM+PB group and the2B1-NRM+PB+PRETREATMENT group showed a decrease in HRP activity overthat of the PB-alone treatment group, indicating an inhibitory effect ofthe 2B1-NRM oligomer on the amount of PB-induced CYP2B1 present in themicrosomal fractions.

Microsomes from rats in the 2B1-ATG+PB treatment group showed asignificant decrease in HRP activity over the PB-alone group, indicatingthat the 2B1-ATG oligomer had a significant inhibitory effect onPB-induced production of CYP2B1, in agreement with the significant sleeptime increases observed in that rat treatment group. Microsomes from2B1-BPD treated rats showed an increase in CYP2B1 over control in theELISA assay, which concurs with the anomalous sleep time reductionobserved in 2B1-BPD treated rats.

1F. Determination of CYP2E1 Induction

Microsomal PNP (p-nitrophenol hydroxylase) activity was used as ameasure of induction of CYP2E1, as PB is known to induce many differentCYPs, including CYP2E1. CYP2E1 activity was determined by PNP activityas described in Reinke and Moyer, Drug Metab. Dispos. 13:548-52, 1985;Koop, Mol. Pharmacol. 29:399-404, 1986). Activity was recorded asoptical density (OD) per milligram of protein per minute.

Microsomes from control rats had a PNP activity of 0.49±0.05 OD/mgprot/min. Microsomes isolated from rat groups treated with PB (PB;2B1-NRM+PB; 2B1-NRM+PB+pretreatment; 2B1-ATG & PB) all showedsignificant increases in PNP activities over that of control,demonstrating induction of CYP2E1 by PB. Since each PB group showedapproximately equivalent PNP activities, the ODNs did not appear tointerfere with the PB induction mechanism of CYP2E1. Microsomes isolatedfrom rats treated with oligomers 2B1-NRM, 2B1-ATG, 2B1-BPD, 2B1-3MM and2B1-CMM (SEQ ID NOs: 17, 16, 36, 37 and 38, respectively) alone (no PB)showed no significant differences in PNP activities from control values.

All data was reported as mean±standard deviation. The mean and standarddeviation were determined by the computer program InStat2 (GraphPad, SanDiego). The p values were also calculated by InStat2 using theTukey-Kramer Multiple Comparisons Test.

Example 2

Antisense Inhibition of Rat Cytochrome p450 (CYP) 2E1

Substrates to cytochrome p450 isozymes frequently control the rate oftheir own metabolism by modulating isozyme gene expression (Eliasson etal., J Biol Chem. 267(22):15765-9, 1992). Cytochrome p450 IIE1 (CYP2E1)up-regulation has been attributed to increased transcription, mRNAstabilization and enhanced stability of the protein. CYP2E1 geneexpression is induced by low molecular weight compounds such as ethanol,actone, and pyrazole.

The exemplary rat CYP2E1 antisense sequences given in Table 2 were usedto evaluate the effectiveness of antisense targeting of specificsequences important to the processes of pre-mRNA splicing and mRNAtranslation.

Oligonucleotides

Phosphorothioate oligonucleotides were synthesized on a 1 μmole scale byuse of an Applied Biosystem Model 380A DNA Synthesizer (Foster City,Calif. and University of Nebraska DNA synthesis core facility),according to standard methodology (e.g. G. Zon and W. J. Stec, inEckstein, F. (ed.), Oligonucleotides and Analogues: A Practical Approach(IRL Press at Oxford University Press), pp. 87-108 (1991). Theoligonucleotides had sequences antisense to rat cytochrome CYP2E1 mRNAand pre-mRNA sequences (Umeno et al., Biochemistry 27(25):9006-13,1988); GenBank Locus RATCYP45I, Accession M20131).

The anti-2E1 30-mer, 5′-(GGT TTA TTA TTA GCT GCA GTT GGC TAT CAT)-3′(SEQ ID NO: 18), is antisense to a region in the rat CYP2E1 sequencebeginning at position 1406 and containing a sequence upstream of the ATGtranslation start site. The sequences of the anti-2E1 20-mers are asfollows: 5′-(CCA AGA ACC GCC ATG GTG CC)-3′ (SEQ ID NO: 19), antisenseto a region beginning at position 1560 and targeting the ATG translationstart site; 5′-(ACC TTG GTG AAA GAC TTG GG)-3′ (SEQ ID NO: 20) antisenseto a region beginning at position 1725 and targeting the splice donor ofexon 1; 5′-(CCT TGT TCT TGT ACT CCT GG)-3′ (SEQ ID NO: 21) antisense toa region beginning at position 2645 and targeting the splice donor ofexon 2; 5′-(GAG AAG CAT GGT CAC CTG GA)-3′ (SEQ ID NO: 22) antisense toa region beginning at position 6681 and targeting the splice donor ofexon 4; and 5′-(CCA ACA CAC ACA CGC TT TCC)-3′ (SEQ ID NO: 23),antisense to a region beginning at position 11591 and targeting thesplice acceptor of exon 9. Two nonsense oligonucleotides prepared forcontrol purposes include the 27-mer 5′-(TCG TCG GTC TCT CCG CTT CTT CCTGCC)-3′ (SEQ ID NO: 41), antisense to rev of HIV-1 (Matsukura et al.,Proc Natl Acad Sci USA 86(11):4244-8, 1989) and the 20-mer 5′-(TCG TGATGA ATT CTG TCG AG)-3′ (SEQ ID NO: 42), with no homologous complementarysequence in the rat genome.

Cell Transfection

The rat hepatoma cell line, H42E, was purchased from American TypeCulture Collection (Bethesda, Md.). The cells were maintained inRPMI-1640 media (Sigma, St. Louis, Mo.), supplemented with 10% heatinactivated fetal bovine serum (Gibco, Grand Island, N.Y.), penicillin G(10,000 units/ml) and streptomycin (10% mg/ml)(Sigma). The cells weresubcultured at 3-4 day intervals at a density of 2×10⁵ cells per 25 cm²flask in 5 ml of medium, and incubated at 37° C. in a humidifiedatmosphere of 95% air, 5% CO₂.

The H42E cells were plated (2×10⁶ to 1.25×10⁵ for the 1 to 5 daytimepoints) in 10 ml RPMI+10% FBS in 100 mm tissue culture dishes(Becton Dickenson, Oxnard, Calif.) and allowed to adhere overnight.Antisense oligonucleotide (3.0 μM) and pyrazole (16 μM; Sigma) wereadded, and dishes were incubated at 37° C. for 1 to 5 days. Cells wereharvested in 2 ml 0.1 M K₂HPO₄ pH 7.2 and homogenized into microsomes.

Eight antisense oligonucleotides, as described above, were employed totest the ability of the various mRNA target regions to inhibitpyrazole-induced synthesis of the CYP2E1 enzyme. Six antisenseoligonucleotides, as described above (SEQ ID NOs: 18-23), were specificto different regions along CYP2E1 mRNA. The last two sequences, MM3 andBUD (SEQ ID NOs: 41 and 42, respectively), were nonsenseoligonucleotides with no targets within the CYP2E1 gene.

The relative concentrations of CYP2E1 enzyme in prepared microsomes weremeasured by use of ELISA. Microsome dilutions of 50 to 6.25 μg/ml ofeach sample were plated in coupling buffer (0.06% sodium carbonate,0.29% sodium bicarbonate, pH 9.6) in 96 well immunoassay NUNC plates(VWR Scientific Chicago, Ill.). Plates were incubated overnight at 4° C.to allow protein to adhere to wells. Wells were washed 10× with 0.05%Tween 20™ (Aldrich, Milwaukee, Wis.)/PBS, the last wash with PBS only,then blocked for 2 hours with 3% BSA (Sigma)/PBS. Wells were washed asbefore. The primary antibody, anti-Cytochrome p450 2E1 (Oxygene, Dallas,Tex.), diluted 1:200 in 1% BSA/PBS, was added and plates were incubatedfor 1.5 hours. Wells were washed, and a 1:2000 dilution of the secondaryantibody, horseradish peroxidase (HRP) conjugate (Bio-Rad, Richmond,Calif.), was added. Plates were incubated 2 hours and washed. A 1:1dilution of ABTS Peroxidase Substrate and Peroxidase Solution B(Kirkegaard and Perry, Gaithersburg, Md.) were added to the wells.Plates were read kinetically at 405 nm every 30 seconds for 1 hour withan OD max of 0.500 (Pruslin, J Immunol Methods 137(1):27-35, 1991).

Results are given in Table 7.

TABLE 7 Characteristics of antisense CYP2E1 oligomers and effect onCYP2E1 enzyme levels SEQ ID ELISA NO: TARGET SITE T_(m) G/C (% ofcontrol) 18 upstream ATG 1406 58.7° C. 36.7% 75.8 ± 11.3^(a) start 19ATG start site 1560 61.9° C. 65.0% 51.7 ± 12.0^(a) 20 exon 1 splice 172550.6° C. 50.0% 66.3 ± 9.7^(a) donor 21 exon 2 splice 2645 46.2° C. 50.0%89.3 ± 6.9 donor 22 exon 4 splice 6681 52.2° C. 55.0% 92.0 ± 7.6 donor23 exon 9 splice 11591 53.7° C. 55.0% 62.9 ± 7.4^(a) accpt 41 nonsensecontrol MM3 70.3° C. 63.0% 99.7 ± 8.7 42 nonsense control BUD 48.1° C.45.0% 95.1 ± 9.0 ^(a)p < 0.05.

Several of the oligonucleotides produced significant reduction in levelsof CYP2E1 enzyme. The antisense oligomers 2E1-1560, 2E1-11591, 2E1-1725and 2E1-1406 (SEQ ID NOs: 19, 23, 20, and 18, respectively) reducedenzyme levels to 52%, 63%, 66% and 76% of control, respectively.Treatment with the other CYP2E1 specific oligonucleotides, as well asthe nonsense oligonucleotides, resulted in CYP2E1 levels that were notsignificantly different from the control level.

Varying doses of the antisense oligonucleotide 2E1-1560 (SEQ ID NO: 19)were investigated for inhibition of the CYP2E1 enzyme in H42E cultures.The cultures showed an increase in inhibition on an increase in oligoconcentration from 0.3 μM to 3 μM, at which the enzyme level wasapproximately 4.1% of control. Above this level, the CYP2E1 proteininhibition leveled off, showing little additional inhibition at 10 μM ofoligonucleotide (FIG. 4).

Example 3

Antisense Inhibition of Rat Cytochrome p450 (CYP) 3A2 by PS and PMOOligonucleotides and Effect on Midazolam Efficacy

Antisense oligonucleotides complementary to target sequences within thecytochrome p450 3A2 (rat CYP3A2) gene sequence were prepared, with theobjective of increasing the effectiveness of midazolam (MZ) in rats. Twophosphorothioate (PS) and two PMO oligomers were synthesized accordingto the known rat CYP3A2 sequence (GenBank Accession No. U09742; seeTable 2).

TABLE 8 Antisense Rat CYP3A2 Oligonucleotide Sequences and Controls RatCYP3A2 mRNA (Accession #U09742):   -8     -1           10         205′-AAGCAGGG AUG GACCUGC UUUCAGCUCU CACACUGG-3′ (SEQ ID NO:45) SEQ ID NO:Name Sequence Type 24 ATG3A2/PS 5′-TGAGAGCTGAAAGCAGGTCCAT-3′ PS DNAAUG3A2/PMO 5′-UGAGAGCUGAAAGCAGGUCCAU-3′ PMO RNA 25 (-3)ATG3A2/5′-GAGMTGAAAGMAGGTM MAT MMM-3′ PMO RNA/C-5 PMO-C5M 43 REV3A2/PS5′-TACCTCGACGAAAGTCGAGAGT-3′ PS DNA; reverse control REV3A2/PMO5′-UACCUCGACGAAAGUCGAGAGU-3′ PMO RNA; reverse control 44 ATGMYC/PS5′-ACGTTGAGGGGCAUCGTCC-3′ PS DNA; myc control AUGMYC/PMO5′-ACGUUGAGGGGCAUCGUCC-3′ PMO RNA; myc control M: 5′-methyl cytidine

The antisense oligomers are shown in Table 8. The oligos designatedAUG3A2/PMO and ATG3A2/PS have the sequence designated SEQ ID NO: 24,which targets the ATG start codon. In (−3)ATG3A2/PMO-C5M (SEQ ID NO: 25,a three-base shift from SEQ ID NO: 24), several cytidine bases are5′-methylated, as shown in the Table. Reverse-sequence oligos (SEQ IDNO: 43) and a c-myc sequence (SEQ ID NO: 44) were used as controls.

Male Sprague-Dawley rats (Sasco, Omaha) weighing between 210 to 290grams were used for all studies. The animals were exposed to 12 hourlight/dark cycle and fed Purina rat chow and tap water ad libitum.

A dose-response curve for MZ was determined by injecting rats i.p. with20, 50 or 70 mg/kg of MZ and recording sleep times (data not shown).Sleep time was defined as the time period from when the rat was placedon its back to when it regained its righting reflex.

Sleep time was measured after i.p. injection of 50 mg/kg MZ (Hoffman-LaRoche, Nutley, N.J.) at 0 (day 1), 24 (day 2) and 48 h (day 4). Thevolume of the injection was from 2.0 to 2.4 ml/rat. All animalsdemonstrated loss of righting reflex within 2 min after i.p. injectionof MZ. The rats were treated with 0.25, 0.50 or 1.00 mg antisense ODNi.p. immediately after the animal had regained its righting reflexdetermined at 0 and 24 h. Control rats received saline only. Totalvolume injection was 0.1 ml in saline.

Preparation of Microsomes

Microsomes were prepared, as described by Franklin and Estabrook (citedabove) for determination of enzyme level and activity. The rats weresacrificed using ethyl ether, and livers were perfused with 12 ml of 4%saline via the portal vein and then removed from the animal. The liverswere minced, homogenized in 0.25 M sucrose (Sigma) and centrifuged at8000×G for 20 minutes at 4° C. in a Sorvall RC2-B centrifuge (Dupont).The supernatant was saved, resuspended in 0.25 M sucrose, andcentrifuged at 100,000×G for 45 minutes at 4° C. in a Sorvall OTD55Bultracentrifuge (Dupont). The pellet was resuspended in 1.15% KCl(Sigma) and centrifuged at 100,000×G for 1 hour at 4° C. The finalpellet was resuspended in an equal volume buffer (10 mM Tris-acetate, 1mM EDTA, 20% glycerol; Sigma) and frozen at −80° C.

Determination of Protein Concentrations

Protein concentrations were determined by Bradford assay (M. M.Bradford, Anal Biochem 72:248-54, 1976). Eighty μl aliquots ofhomogenate, prepared as described above, were added to a 96 well plate(Becton Dickinson Labware, Lincoln Park, N.J.). Twenty μl of Bradfordreagent (Bio-Rad Richmond, Calif.) was then added and the plates read at595 nm on the microplate reader (Molecular Devices, Newport Minn.). Thedata was compared to standard curve generated with known concentrationsof bovine serum albumin (Sigma).

Liver Microsomal Assays for CYP3A2

Erythromycin demethylation (ED) was used a measure of CYP3A2 enzymaticactivity (Gonzalez, Pharmacol. Rev. 40:243-87, 1989). Activity wasrecorded as micromoles of formaldehyde per milligram of protein perminute.

Western blot of CYP3A2

Western blot analysis of CYP3A2 was carried out using the methoddescribed by Tracewell et al., Toxicol Appl Pharmacol. 135(2):179-84,1995. Band intensities were determined by a Molecular Dynamics PersonalDensitometer (Sunnyvale, Calif.) with ImageQuant version 3.3 software(Molecular Dynamics).

Statistical Analysis

All microsomal data were reported as mean standard error of the mean(S.E.) as determined by the computer program InStat2 (Graphpad, SanDiego). The P values were also calculated by InStat2 with the Tukeymultiple comparison test. Standard curve and graphs were generated usingPrism (GraphPad).

Results

Sleep Time

Table 9 shows the change in sleep time (MZ ST) and ED activity (a markerfor CYP3A2) for animals receiving the various treatments. Both the PMOand PS oligos targeting the start codon (SEQ ID NOs: 24 and 25) showedan increase in sleep time, as would result from inhibition of CYP3A2 andconsequent inhibition of metabolism of the MZ. The PMO oligo, however,was effective at one-tenth the concentration of the PS oligo. Controloligos (SEQ ID NOs: 43 and 44) showed little or no change from thesaline-only control.

ED Activity

All of the anti-3A2 oligos (SEQ ID NOs: 24 and 25) reduced ED activityto some extent, with the 5′-C-methyl modified PMO (SEQ ID NO: 25)showing greater activity than the unmodified oligo. Activity in animalsadministered control oligos was not significantly different from thesaline control.

TABLE 9 Effect of Anti-CYP3A2 Oligonucleotides on MZ Sleep Time and EDActivity in Rats SEQ Treatment ID Dose (no. of animals) NO: (μg/day) MZST ED Saline control (10) — — 22.3 ± 0.9 100 ± 3.3 ATG3A2/PS 24 100035.3 ± IS 52 ± 13^(a) AUG3A2/PMO (4) 24 100 33.3 ± 2.3^(a) 80 ± 5^(b)(−3)ATG3A2/PMO-C5M (4) 25 100 nd 55 REV3A2/PS (3) 43 1000 22.4 ± 0.6 91REV3A2/PMO (3) 43 100 20.3 ± 1.3 99 ATGMYC/PS (4) 44 1000 22.8 ± 1.3 113AUGMYC/PMO (3) 44 100 20.6 ± 0.6 110 All values represent the mean ±standard error of the mean. MZ ST — midazolam sleep time, min; ED —erythromycin demethylase activity (in vitro marker for CYP3A2), μmolHCOH/mg/min. ^(a)Significantly different from saline, myc, and revgroups, p < 0.01. ^(b)Significantly different from myc and salinegroups, p < 0.05.

V79 cells were stably transfected with the human CYP3A4 gene. The cellswere scrape loaded with 10 uM PMO having the sequences shown below,targeting the ATG start codon of human CYP3A4 mRNA (SEQ ID NOs: 46, 47,and 35; see Table 2). A sequence targeting the ATG rat CYP3A2 (SEQ IDNO: 25, with C-methyl substitution) was also employed. Activity wasassayed in S-9 fractions via 7-benzyloxy-4-(trifluoromethyl)-coumarinconversion to fluorescent product7-hydroxy-4-(trifluoromethyl)-coumarin, a CYP3A4 specific reaction.

    5′-CTG GGA TGA GAG CCA TCA C-3′ SEQ ID NO: 46 human CYP3A4    5′-CTG GGA TGA GAG CCA TCA CT-3′ SEQ ID NO: 47 "  5′-GT CTG GGA TGAGAG CCA TCA C-3′ SEQ ID NO: 35 " 5′-GAG MTG AAA GMA GGT MMA TMM M-3′ SEQID NO: 25 rat CYP3A2

pmoles/50 μg protein Treatment in 10 minutes Vehicle (control) 23.80Scrambled control oligo 21.60 SEQ ID NO:25 21.90 SEQ ID NO:46 13.10 SEQID NO:47 13.40 SEQ ID NO:35 16.50

As shown in the table, the human antisense sequences significantlyreduced enzyme activity. The anti-rat oligo, which in this case has onlyabout 55% homology with the human sequence targeted, showed no reductionin enzyme activity.

Example 5

Effectiveness of Orally Administered PMO Antisense Oligomers

FIG. 7 shows a Western blot of liver microsome samples obtained fromrats which were administered a phosphorodiamidate morpholinooligonucleotide (PMO) antisense to the rat CYP3A2 gene (SEQ ID NO: 25,with no C-methyl modifications), either i.p. or orally. The blot wasfirst probed with anti-rat CYP3A2 antibodies. After stripping offantibodies, the blot was re-probed with antibodies to NADPH Reductase asa control for total protein loading in the various lanes.

Each rat weighed approximately 200 gm, and was treated 24 hours prior toorgan harvesting with the following: saline, injected intraperitoneally(lane 1); 15 nmoles of PMO 1-0-328, injected intraperitoneally (lanes 2and 3); 60 nmoles of PMO 1-0-328, administered orally (lanes 4 and 5).Each a different test animal.

No significant reduction in CYP3A2 protein compared to thesaline-injected control was observed 24 hours after i.p. injection of 15nmoles (approx. 0.5 mg/kg body weight) PMO (lanes 2-3); however, inother experiments, a modest decrease in CYP3A2 enzyme activity wasobserved under these conditions. Furthermore, a significant reduction inCYP3A2 protein level was observed by Western blot when a secondinjection of 15 nmoles PMO was administered 24 hours following thefirst, and organs were harvested 24 hours thereafter (data not shown).

In a test of the oral bioavailability of antisense PMOs, 60 nmoles ofthe antisense PMO (2 mg/kg body weight, four times the i.p dose usedabove) were administered to rats by oral gavage, and organs wereharvested 24 hours later. Lanes 4 and 5 show a significant reduction inCYP3A2 protein compared to lanes 2 and 3, showing that the relative oralbioavailability of the antisense PMO is substantially greater than 25%of the i.p. administered PMO.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

47 1 33 DNA Rat 1 gacagacaag cagggatgga cctgctttca gct 33 2 33 DNA Mouse2 gacagacaag cagagatgaa cctattttca gcg 33 3 33 DNA Mouse 3 ttaaagaaaacagcaatgga cctgatccca aac 33 4 33 DNA Mouse 4 gacaaacaag cagggatggacctggttttc agc 33 5 33 DNA Hamster 5 aaatcgcaca aggaaatgga cctggtccccagc 33 6 33 DNA Rabbit 6 agaaggacag tggcgatgga tctgatcttt tcc 33 7 33DNA Dog 7 agaggacgag tggtcatgga cttcatccca agc 33 8 33 DNA Pig 8acgaggacag tggccatgga cctgatccca ggc 33 9 33 DNA Goat 9 gccaagaaagtggccatgga gctgatccca agt 33 10 33 DNA Monkey 10 ggaaggaaag tagtgatggatctcatccca gac 33 11 33 DNA Human 11 gtaaggaaag tagtgatggc tctcatcccagac 33 12 33 DNA Human 12 gtaaggaaag tagtgatggc tctcatccca gac 33 13 33DNA Human 13 agaaggaaag tggcgatgga cctcatccca aat 33 14 33 DNA Human 14agaaggcaag tggcgatgga cctcatccca aat 33 15 21 DNA Human 15 gtgatggatctcatcccaaa c 21 16 22 DNA Artificial Sequence antisense 16 ggagcaagatactgggctcc at 22 17 22 DNA Artificial Sequence antisense 17 aaagaagagagagagcaggg ag 22 18 30 DNA Artificial Sequence antisense 18 ggtttattattagctgcagt tggctatcat 30 19 20 DNA Artificial Sequence antisense 19ccaagaaccg ccatggtgcc 20 20 20 DNA Artificial Sequence antisense 20accttggtga aagacttggg 20 21 20 DNA Artificial Sequence antisense 21ccttgttctt gtactcctgg 20 22 20 DNA Artificial Sequence antisense 22gagaagcatg gtcacctgga 20 23 20 DNA Artificial Sequence antisense 23ccaacacaca cacgctttcc 20 24 22 DNA Artificial Sequence antisense 24tgagagctga aagcaggtcc at 22 25 22 DNA Artificial Sequence antisense 25gagctgaaag caggtccatc cc 22 26 20 DNA Artificial Sequence antisense 26attgggaaaa gcatgatcag 20 27 20 DNA Artificial Sequence antisense 27tgggacaatg ccatctgtac 20 28 20 DNA Artificial Sequence antisense 28aggctggtgc ccatgctgcg 20 29 20 DNA Artificial Sequence antisense 29cctgaggcca gcatggtggt 20 30 20 DNA Artificial Sequence antisense 30acgctgagtt ccatggtctg 20 31 20 DNA Artificial Sequence antisense 31acaagagaat ccattgaagc 20 32 20 DNA Artificial Sequence antisense 32cacaaaagga tccattgaag 20 33 20 DNA Artificial Sequence antisense 33gcttctagcc ccatacctgc 20 34 20 DNA Artificial Sequence antisense 34ccgagggcag acatggtgcc 20 35 21 DNA Artificial Sequence antisense 35gtctgggatg agagccatca c 21 36 20 DNA Artificial Sequence antisense 36aaagaagaga gagcagggag 20 37 20 DNA Artificial Sequence antisense 37aaagaagaga gagcagggga 20 38 20 DNA Artificial Sequence antisense 38aaagaagaga aggcagggag 20 39 28 DNA Rat misc_feature (0)...(0) 3′ to 5′39 cctcctcgtt ctatgacccg aggtacca 28 40 28 DNA Rat misc_feature(0)...(0) 3′ to 5′ 40 tcgtttcttc tctctctcgt ccctctag 28 41 27 DNAArtificial Sequence oligonucleotide 41 tcgtcggtct ctccgcttct tcctgcc 2742 20 DNA Artificial Sequence oligonucleotide 42 tcgtgatgaa ttctgtcgag20 43 22 DNA Artificial Sequence reverse control 43 tacctcgacgaaagtcgaga gt 22 44 19 DNA Artificial Sequence myc control 44 acgttgaggggcaucgtcc 19 45 36 RNA Rat 45 aagcagggau ggaccugcuu ucagcucuca cacugg 3646 19 DNA Artificial Sequence antisense 46 ctgggatgag agccatcac 19 47 20DNA Artificial Sequence antisense 47 ctgggatgag agccatcact 20

It is claimed:
 1. A method of inhibiting metabolism of a drugadministered to a mammalian subject, comprising co-administering withsaid drug a morpholino antisense oligomer effective to reduce expressionof a cytochrome p450 enzyme that catalyzes metabolism of the drug insaid subject, by hybridizing to a target RNA molecule which encodes saidenzyme; wherein the cytochrome p450 is selected from the groupconsisting of CYP2B1, CYP2E1, CYP3A2, and CYP3A4; and wherein saidmorpholino oligomer: is composed of morpholino subunits, linked togetherby uncharged phosphorus-containing linkages, one to three atoms long,joining the morpholino nitrogen of one subunit to the 5′ exocycliccarbon of an adjacent subunit, and includes, linked to each subunit, apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide, and has abase sequence selected from the group consisting of: SEQ ID NO: 16, forinhibition of CYP2B1, SEQ ID NOs: 18-20 and 23, for inhibition ofCYP2E1, SEQ ID NOs: 24 and 25, for inhibition of CYP3A2, and SEQ ID NOs:35, 46, and 47, for inhibition of CYP3A4.
 2. The method of claim 1,wherein the drug either induces said drug-metabolizing cytochrome p450enzyme, or is administered to a subject who has been exposed to axenobiotic agent which induces such an enzyme.
 3. The method of claim 2,wherein said drug induces at least one cytochrome p450.
 4. The method ofclaim 2, wherein said xenobiotic agent induces at least one cytochromep450.
 5. The method of claim 1, wherein the antisense oligomer has anuncharged backbone comprising phosphoramidate or phosphorodiamidatelinkages.
 6. The method of claim 1, wherein the antisense oligomerhybridizes to a region of said target RNA with a T_(m) greater than 37°C.
 7. The method of claim 1, wherein the antisense oligomer isadministered orally to the subject.
 8. The method of claim 7, whereinsaid oligomer is administered in an amount of at least 1 mg/kg bodyweight.
 9. The method of claim 3, wherein said cytochrome p450 isCYP2E1, and said drug is acetaminophen.
 10. The method of claim 3,wherein said cytochrome p450 is the CYP2B1 or CYP3A4, and said drug isphenobarbital or hexobarbital.
 11. The method of claim 10, wherein saidcytochrome p450 is CYP2B1.
 12. The method of claim 3, wherein saidcytochrome p450 is CYP3A4, and said drug is an antibiotic selected fromthe group consisting of clarithromycin, erythromycin, rifampicin,rifampin, rifabutin, and rapamycin.
 13. The method of claim 3, whereinsaid cytochrome p450 is CYP3A4, and said drug contains an estrogen orestradiol.
 14. The method of claim 4, wherein said cytochrome p450 isCYP3A4, said drug is a protease inhibitor or non-nucleoside reversetranscriptase inhibitor, and said xenobiotic is a CYP3A4-inducingnon-nucleoside reverse transcriptase inhibitor.
 15. The method of claim1, wherein said cytochrome p450 is CYP3A4, and the drug is paclitaxel.16. The method of claim 5, wherein the morpholino oligomer is composedof morpholino subunits joined by phosphoramidate or phosphorodiamidatelinkages, in accordance with the structure:

where Y₁ is oxygen, Z is oxygen, X is alkoxy, amino, or dialkyl amino,and Pj is a purine or pyrimidine base-pairing moiety effective to bind,by base-specific hydrogen bonding, to a base in a polynucleotide. 17.The method of claim 1, wherein the cytochrome p450 is CYP2B1, and themorpholino oligomer has a base sequence selected from SEQ ID NO:
 16. 18.The method of claim 1, wherein the cytochrome p450 is CYP2E1, and themorpholino oligomer has a base sequence selected from SEQ ID NOs: 18-20and
 23. 19. The method of claim 1, wherein the cytochrome p450 isCYP3A2, and the morpholino oligomer a the base sequence selected fromSEQ ID NOs: 24 and
 25. 20. The method of claim 1, wherein the cytochromep450 is CYP3A4, and the morpholino oligomer has a the base sequenceselected from SEQ ID NOs: 35, 46, and 47.